Our previous studies indicated that release of preformed granules from cultured RBL-2H3 mast cells is dependent on protein kinase C (PKC) whereas release of arachidonic acid is dependent on MAP kinase for the phosphorylation and activation of phospholipase (PL) A2. Both processes are dependent on an increase in calcium for activation of secretory machinery and translocation of the phosphorylated PLA2 to membranes (see report ZO1 HL 00990-10 LMI for description of regulation of cytokine release in RBL-2H3 cells). In the case of antigen-stimulated cells, these processes are mediated through recruitment of the tyrosine kinases, Lyn and Syk. As further elaboration of this scenario, we now show that activation of PKC (and secretion) is dependent on the activation of a membrane-associated cholera-toxin-sensitive PLD when cells are stimulated with the Ca2+-mobilizing agents, calcium ionophore and thapsigargin, and dependent in part on this PLD activity when cells are stimulated via receptors. As demonstrated in intact and cell-free systems, the toxin-sensitive PLD can be activated by PKC, elevated free calcium or beta/gamma subunits of trimeric G proteins and is regulated by phosphatidylinositol 3-kinase. These properties suggest that the toxin-sensitive PLD differs from the recently described ARF-regulated Golgi-associated PLD. Consistent with this scenario, a variety of secretagogues stimulated PLD and secretion and both responses were equally enhanced by treatment with cholera toxin. Both responses were suppressed by the phosphatidylinositol 3-kinase inhibitor, wortmannin; the PLD/PKC inhibitor, butanol; the phosphatidate phosphohydrolase/PKC inhibitor, propranolol; and by the PKC inhibitor, Ro31-7549. With repect to MAP kinase, we now find that both p38 and p42 MAP kinases are activated in antigen-stimulated cells but only the latter kinase appears to be essential for release of arachidonic acid (as indicated by selective inhibitors). The p42 MAP kinase, contrary to published reports, is activated synergistically via calcium and PKC in addition to a third pathway through Shc, Grb2, Sos and Ras. Interestingly, antigen stimulates p42 MAP kinase transiently via PKC and then via a long-lived signal, possibly the aforementioned third pathway. Finally, with respect to distal events, there is accumulating information on essential roles for G proteins and other regulatory proteins in RBL-2H3 cells. For example, activators of G proteins such as guanine nucleotides or the mast cell activator, compound 48/80, which is thought to act via the trimeric G protein Gi3 (see last years report), can stimulate secretion independently of PKC or a rise in calcium (see also report ZO1 HL 00993-10 LMI).